Protocol for multi-modal single-cell RNA sequencing on M. tuberculosis-infected mouse lungs

Summary To elucidate how different immune cells contribute to control or progression of M. tuberculosis (Mtb) infection, we developed a technique to perform multi-modal single-cell RNA sequencing (scRNA-seq) from in vivo Mtb-infected lung macrophages. This protocol simultaneously acquires the transcriptome, surface marker expression, and bacterial phenotype of each infected cell. We describe steps for sorting Mtb-infected cells and staining with CITE-seq antibodies, as well as for methanol fixation and generation of scRNA-seq libraries. This protocol can be used on tissues derived from murine, nonhuman primate, and human infections. For complete details on the use and execution of this protocol, please refer to Pisu et al. (2021).1


MATERIALS AND EQUIPMENT
Alternatives: this protocol describes the generation of a single-cell lung suspension using a GentleMACS (with heaters) instrument from Miltenyi Biotec. If a GentleMACS is not available, it is possible to generate a single-cell suspension manually as described in previous protocols. 4,5 Alternatives: this protocol was originally performed using the 103 Single Index Next GEM 3 0 reagent kit v 3.1 (protocol CG000206 Rev D.), which will be discontinued starting December 2022. A protocol that makes use of HTO and ADT primers compatible with dual indexing is available from Biolegend.
Alternatives: this protocol requires the use of a Qubit instrument to measure the concentration (in ng/mL) of the HTO and ADT libraries. If a Qubit is not available, it is possible to submit the libraries for analysis with a Fragment Analyzer before sequencing to check both the quantity and the quality of the resulting DNA.
Alternatives: in this protocol we perform HTO and ADT staining before methanol fixation. However, we successfully tested the staining of methanol-fixed samples with both HTO and ADT antibodies, therefore enabling multi-modal scRNA-seq from clinical samples, fixed at the time of collection. See later part of the protocol for detailed step-by-step instructions. Alternatives: here, we make use of TotalSeq-A antibodies from Biolegend (which contain a poly(A) tail and are compatible with any sequencing platform that relies on poly(dT) for transcript capture). However, we successfully used TotalSeq-B antibodies whose capture sequence is specific for the 103 single cell 3 0 kits (v3 and v3.1). We first autoclave a solution containing 7H10 powder, Glycerol and MilliQ water for 15'. After cooling down, we add OADC enrichment and Cycloheximide in a sterile hood. We then pour the 7H10 agar directly into Petri dishes, that are then stored at 20 C-25 C until use. We don't store 7H10 plates long-term. We typically use the prepared 7H10 plates in 7-10 days. We filter-sterilize the washing solution and store it at 4 C. We don't store the washing solution long-term. We typically use the prepared washing solution in 7-10 days. In this step we prepare, freeze and titer the bacterial stocks needed for mice infection.

Reagent
1. Grow an aliquot of each bacterial strain needed for the experiment in a 37 C incubator, using 10 mL of 7H9 media in a 25 cm 2 polystyrene tissue culture flask with vented cap, up to log phase (OD 600 = 0.6-0.8).
CRITICAL: make sure to add the appropriate antibiotic selection marker for your strain. For our experiment we grew the hspx'::GFP/smyc'::mCherry strain in 7H9 media + 50 mg/mL of Hygromycin B. Failure to add the selection marker will result in loss of plasmid and relative fluorescence during mouse infection, and consequently it won't be possible to discriminate between infected vs bystander cells during cell sorting.
2. After $ 7 days of growth (depending on the initial inoculum), check OD 600 with a spectrophotometer. If bacteria have reached log phase go to step 3, otherwise keep growing them until they reach OD 600 = 0.6-0.8. 3. Once bacteria have reached log phase, spin down the culture at 2,800 3 g for 10 0 at 20 C-25 C, using a 15 mL falcon tube.

Reagent Final concentration (mM or mM) Amount
Washing Solution N/A 3.98 mL Collagenase IV 250 U/mL 20 mL

Total N/A 4 mL
We make stocks of Collagenase IV at 50,000 U/mL and store it at À20 C until use. The maximum recommended storage time is 1 year. Right before collecting mouse lungs, we prepare the Dissociation Solution adding Washing Solution + Collagenase IV directly into the GentleMacs C tubes under sterile conditions. We prepare the Sorting Buffer the day before sorting. We filter-sterilize the sorting buffer and store it at 4 C until use. We don't store the sorting buffer long-term. We always prepare fresh sorting buffer.

Reagent Final concentration (mM or mM) Amount
RNAse inhibitor 0.5 U/mL 2 5 mL We prepare the Rehydration Buffer fresh the day of the experiment and keep it on ice until needed. 4. While the bacterial cultures are spinning down, prepare and mark the amount of freezing vials that are needed to stock and freeze the bacterial samples (20 vials for each 10 mL culture). 5. Resuspend the bacterial pellet in 1 mL of 7H9 media plus antibiotic (if appropriate). 6. Using a 1 mL tuberculin syringe with a 25 gauge needle insert the syringe into the falcon tube and pass the bacterial culture in and out of the syringe for 20 times.
CRITICAL: proceed to the next step immediately, so that bacteria won't have time to clump.
7. Add 9 mL of 7H9 media plus antibiotic (if appropriate) and slowly pipette the bacterial culture up and down 10 times. 8. Immediately transfer 500 mL of culture to each freezing vial. 9. Add 500 mL of a 40% glycerol stock solution to each freezing vial containing the bacterial culture (final volume = 1 mL) and pipette up and down until the bacterial stock solution appears homogenous. 10. Store the bacterial stock solutions at À80 C for 7 days. 11. After 7 days, thaw 3 randomly selected frozen vials at 20 C-25 C.
Note: we wait 7 days at À80 C because after freezing some bacteria will die and we want to precisely determine the titer of the bacterial stocks prior to mice infection.
12. Transfer each bacterial stock to a 15 mL tube and passage it through a BD tuberculin syringe (25G needle) 15 times to breakup bacterial clumps. 13. Transfer 200 mL of each bacterial stock to a 96-well plate to perform serial dilutions and then plate for colony forming units (CFU) on 7H10 agar plates. 14. After 21 days, count the number of bacterial colonies to determine the titer of the frozen bacterial stocks.

Mouse infection
Timing: $ 2 h In this step of the protocol we are going to infect mice intranasally with 1.5 3 10 3 CFU of our bacterial stocks.
15. Thaw the bacterial aliquots that are needed for mice infection at 20 C-25 C.
Note: for cell sorting of the infected and bystander populations we infect some extra mice to use as gating and compensation controls. In the case of, 1 we used a mouse infected with hsp60'::GFP Erdman (bacteria that constitutively express GFP), a mouse infected with smyc'::mCherry Erdman (bacteria that constitutively express mCherry) and a mouse infected with WT Erdman Mtb to be used as a compensation control for CD45 staining (See Figures S1A-S1C of 1 ).
16. Transfer the bacterial cultures into 15 mL falcon tubes and passage them through a 1 mL BD Tuberculin Syringe with a 25G needle 15 times to breakup bacterial clumps. 17. Depending on the titer of the bacterial stocks calculated on step 14, calculate the number of dilutions to reach a final concentration of 5 3 10 4 bacteria/mL needed for mice infection. Perform the dilutions in infection buffer, using 2 mL Eppendorf screw cap tubes.
CRITICAL: slowly pipette up and down 5-6 times when you transfer Mtb in the subsequent dilution tube to allow homogenous resuspension of the bacteria in the tube and avoid carrying over potential clumps.

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18. Anesthetize the mice using an isoflurane and oxygen mixture (5% isoflurane in oxygen at 4.5 L/min; VIP 3000 isoflurane vaporizer) for $ 2 min, until the animals are sedated and the breath slows down. 19. 30 mL (1.5 3 10 3 bacteria) are then administered intranasally to each mouse.
Note: mice infected with different strains are kept in different cages for the duration of the infection, to prevent strain cross-contamination. We suggest preparing and labeling the cages before performing infection to avoid any potential mix-up when working with high numbers of animals.
Generation of a single-cell suspension and antibody staining for flow sorting Optional: resuspend in 1 mL of Fc blocking solution (Washing solution + Fc block (anti CD16/ 32 for mouse)) and incubate for 15 0 at 4 C. Spin down at 500 3 g for 3 0 at 20 C-25 C.
Note: this step is only necessary if sorting using surface markers staining to discriminate and gate on specific immune populations. For our single-cell RNA-seq paper, we sorted directly on bacterial fluorescence to discriminate infected cells and we only used CD45 to sort the Bystander population, therefore we didn't perform blocking.  Note: we fix the cells at this stage, since the subsequent steps involving library generation and sequencing are performed outside the BSL3.
48. Store at À20 C for up to a week or at À80 C for long term storage. We typically resuspend in 40 mL of Rehydration Buffer for every 60,000 sorted cells. Keep on ice. 57. Count the cells for each sample with a hemocytometer, withdrawing 10 mL volume from each sample. 58. Adjust the concentration of each sample to be between 700-1,200 cells/mL using Rehydration Buffer (this is the recommended input requirement for loading into the 103 chip).

Rehydration of the fixed cells
Note: at this point samples tagged with different HTOs can be combined in the desired proportions to multiplex into a single 103 run. If HTOs have not been used, then every sample needs to be run separately into a single 103 run.
Alternative approach: HTO and ADT staining after methanol-fixation.
a. Equilibrate methanol-fixed samples on ice for 15'. b. In the meantime, prepare HTO and ADT antibody pools as described above, using Cell Staining Buffer (Biolegend) containing 0.5 U/mL of RNase inhibitor. c. Spin down samples at 500 3 g, 4 C for 10'.

Generation of CITE-seq libraries and sequencing
To generate CITE-seq libraries we follow the commercially available protocols from 103 (CG000206 Rev D) with the following modifications.
59. In step 1.1: we use the single-cell suspension generated in step 58 of the ''rehydration of the fixed cells'' section as the input for step 1.2b of the 103 protocol.
CRITICAL: first add the appropriate amount of Nuclease Free Water to the master mix and pipette mix 15 times, until the solution is homogeneous. Do not add nuclease free water to the single cell suspension.
CRITICAL: Pipette mix the single cell suspension 20 times before adding to the master mix, to breakup any potential clumps that could clog the chip and lead to a failed run.

EXPECTED OUTCOMES
Upon execution of the above protocol, the user will generate 3 different libraries (mRNA, HTO, ADT) each containing different indexes that will be pooled and sequenced in a single sequencing run.
We always pool the libraries such that at least 85% of all reads belong to the mRNA library. If using % 20 ADT/HTO markers, we pool libraries using the following proportions: 90% mRNA, 5% ADT, 5% HTO; if using > 20 ADT/HTO markers we pool using the following proportions: 85% mRNA, 10% ADT, 5% HTO.
We allocate an entire Nextseq 500 flow cell ($400 M reads) for each 103 sample containing 10,000 cells. When pooling multiple 103 samples in a single sequencing run we either use an entire NextSeq 2k or Novaseq S4 lane, depending on the amount of samples and desired reads output.
Before sequencing, quality of the generated libraries should be assessed by Fragment Analyzer. Users should expect to see a distribution of fragments for the mRNA libraries (300-600 bp as described in the 103 commercially available protocol CG000206 Rev D) with only minor peaks under 200 bp. For the HTO and ADT libraries users should expect to observe a single peak centered at $210 bp.
The Mtb-infected lung samples contain high proportions of different macrophage populations: we typically recover an average of 800-1,200 genes/cell using the methanol-fixation protocol illustrated above.
After data analysis, users should be able to recover and clearly separate different populations of alveolar and interstitial macrophages.

LIMITATIONS
This protocol has been developed to use with the 103 scRNA-seq 3 0 Gene Expression kits. We have not tested whether our protocol is compatible with the 5 0 scRNA-seq immune repertoire kits from 103. Furthermore, our protocol is not compatible with scATAC-seq because of the methanol fixation step, which will degrade the chromatin organization structure of the nuclei.
Our protocol involves fluorescent activated cell sorting of live material to isolate infected from bystander cells, which may not be an available option in all laboratories. Finally, our protocol requires the use of fluorescent reporter strains to sort infected cells based on the fitness of the bacterial pathogen, which while relatively available for M. tuberculosis, may not be available for other infectious agents.

Problem 1
Low number of cells recovered for input into the 103 chip in step 58 of the ''rehydration of the fixed cells'' section.

Potential solution
Increase the number of sorted cells. We found 60,000 sorted cells to be a reasonable compromise with respect to the duration of sorting time and number of cells that are recovered at the end of the protocol for input into the 103 chip. Be careful not to disturb the pellet during the washing steps of the ''Methanol fixation'' and ''rehydration of the fixed cells'' parts of the protocol. The pellet generated from 60,000 cells will be visible at the bottom of the 1.5 mL Eppendorf tubes. Disturbing the pellet during the cell wash steps will lead to decreased cell recovery.

Problem 2
Failed 103 run resulting in a wetting failure or reagent clog.

Potential solution
Make sure to resuspend the single-cell suspension pipetting up and down at least 20 times before adding to the Master mix. We also suggest resuspending the single cell suspension plus master mix at least 15 times before loading in the 103 chip. Make sure no bubbles are present into Row 1 and Row 2 of the 103 chip after loading of the reagents, before inserting the chip into the controller. Make sure to not touch the bottom of the 103 chip, to avoid creating static electricity which can result in a failed run.

Problem 3
High amount of background RNA among the samples, which will lead to low numbers of genes/cell detected, high number of empty ''cells'' and <30% reads matching the reference sequence.

Potential solution
Make sure that your infection model does not induce extensive necrosis among your samples. We tried sorting samples from RAG1 KO mice, which 3 weeks post Mtb infection had high amounts of necrotic tissue, and we found that elevated levels of background RNA were present in the samples, resulting in unusable 103 runs. Try to avoid tissues containing large numbers of cells with low RNA content, such as neutrophils, which will lyse during the execution of this protocol, resulting in release of background RNA in the samples.
Thoroughly clean the sorter before use and let a solution of RNAse zap run through the sample line for 5 0 , to avoid carryover of background RNA and RNAse (which will degrade your samples) from previous usage.

Problem 4
Low/high yields of the mRNA libraries < 1 ng or > 200 ng.

Potential solution
Adjust the PCR cycle numbers in the ''cDNA amplification'' step 2.2 and ''Sample Index PCR'' step 3.5 of the 103 manual. Make sure to increase/decrease the amount of PCR cycles depending on the type of immune cells that are prevalent in your sample (high/intermediate/low RNA content). Make sure to count and to load the correct number of cells in the master mix step 1.2 of the 103 manual. For example, if we target to recover 10,000 cells, but we only load 4,000 in the chip and we setup the number of PCR cycles in the cDNA amplification step 2.2 such as we loaded 10,000 cells, the resulting yields for the mRNA libraries will be low. Make sure that > 80% of the cells in your sorted samples are viable at the time of methanol fixation.

Problem 5
Low complexity of the mRNA libraries.

Potential solution
Adjust the PCR cycle numbers in the ''cDNA amplification'' step 2.2 of the 103 manual to avoid overamplification of specific cDNA sequences.

Problem 6
Presence of a significant peak < 100 bp in the HTO, ADT and mRNA libraries.

Potential solution
This is usually due to carryover of adapter primers in the sequencing reaction. Perform a 13 SPRI beads cleanup on the mRNA library and/or a 1.23 SPRI beads cleanup on the ADT and HTO libraries, to remove any peak < 100 bp.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, David G. Russell (dgr8@cornell.edu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
The raw datasets needed to repeat the analysis published in J Exp Med are available on GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE167232.

ACKNOWLEDGMENTS
The work was supported by grants AI134183, AI155319, and AI136097 to D.G.R. from the National Institutes of Health and the Bill and Melinda Gates Foundation (OPP1108452 to D.G.R.).

AUTHOR CONTRIBUTIONS
D.P. designed and executed the protocol. D.P. and D.G.R. drafted and edited the manuscript.

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